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pHAGE-TO-nmdCas9-3XGFP

Lentiviral vector for labeling chromosomal loci by tetracycline-regulatable expression of catalytically dead Neisseria meningitidis dCas9 fused to three copies of GFP.

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pHAGE-TO-nmdCas9-3XGFP Sequence and MappHAGE-TO-nmdCas9-3XGFP.dna
Map and Sequence File   
Sequence Author:  Pederson Lab / Addgene #64109
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 AvrII (11,672) PciI (11,393) DrdI (11,291) AhdI (10,505) PvuI (10,136) SspI (9700) PacI (9490) SmaI (9482) TspMI - XmaI (9480) PsiI (9443) XbaI (8505) PaeR7I - PspXI - XhoI (8491) BspDI - ClaI (7766) NotI (7032) BamHI (6301) SpeI (6268) NruI * (833) FseI (1150) MfeI (1189) KflI (1934) NheI (2221) BmtI (2225) PspOMI (2233) ApaI (2237) SnaBI (2578) CMV promoter tet operator tet operator SalI - SgrDI (2850) BsmBI (2881) AgeI (2944) ATG SV40 NLS EcoRI (3007) MreI - SgrAI (3233) SbfI (3641) PasI (5339) Bsu36I (5468) BmgBI (5868) pHAGE-TO-nmdCas9-3XGFP 12,122 bp
AvrII  (11,672)
1 site
C C T A G G G G A T C C
PciI  (11,393)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (11,291)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
AhdI  (10,505)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
PvuI  (10,136)
1 site
C G A T C G G C T A G C
SspI  (9700)
1 site
A A T A T T T T A T A A
PacI  (9490)
1 site
T T A A T T A A A A T T A A T T
SmaI  (9482)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (9480)
1 site
C C C G G G G G G C C C
XmaI  (9480)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
PsiI  (9443)
1 site
T T A T A A A A T A T T
XbaI  (8505)
1 site
T C T A G A A G A T C T
PaeR7I  (8491)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (8491)
1 site
V C T C G A G B B G A G C T C V
XhoI  (8491)
1 site
C T C G A G G A G C T C
BspDI  (7766)
1 site
A T C G A T T A G C T A
ClaI  (7766)
1 site
A T C G A T T A G C T A
NotI  (7032)
1 site
G C G G C C G C C G C C G G C G
BamHI  (6301)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can
remain bound to DNA and alter its electrophoretic mobility.
SpeI  (6268)
1 site
A C T A G T T G A T C A
NruI  (833)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
FseI  (1150)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
MfeI  (1189)
1 site
C A A T T G G T T A A C
KflI  (1934)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
NheI  (2221)
1 site
G C T A G C C G A T C G
BmtI  (2225)
1 site
G C T A G C C G A T C G
PspOMI  (2233)
1 site
G G G C C C C C C G G G
ApaI  (2237)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
SnaBI  (2578)
1 site
T A C G T A A T G C A T
SalI  (2850)
1 site
G T C G A C C A G C T G
SgrDI  (2850)
1 site
C G T C G A C G G C A G C T G C
BsmBI  (2881)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
AgeI  (2944)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
EcoRI  (3007)
1 site
G A A T T C C T T A A G
MreI  (3233)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (3233)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
SbfI  (3641)
1 site
C C T G C A G G G G A C G T C C
PasI  (5339)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
Bsu36I  (5468)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BmgBI  (5868)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
NmdCas9
3019 .. 6261  =  3243 bp
1081 amino acids  =  124.0 kDa
   Segment 1:  
   3019 .. 3060  =  42 bp
   14 amino acids  =  1.5 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Neisseria meningitidis Type
II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D16A,
D587A, H588A, and N611A
NmdCas9
3019 .. 6261  =  3243 bp
1081 amino acids  =  124.0 kDa
   Segment 2:  
   3061 .. 3063  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Neisseria meningitidis Type
II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D16A,
D587A, H588A, and N611A
NmdCas9
3019 .. 6261  =  3243 bp
1081 amino acids  =  124.0 kDa
   Segment 3:  
   3064 .. 4773  =  1710 bp
   570 amino acids  =  65.1 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Neisseria meningitidis Type
II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D16A,
D587A, H588A, and N611A
NmdCas9
3019 .. 6261  =  3243 bp
1081 amino acids  =  124.0 kDa
   Segment 4:  
   4774 .. 4779  =  6 bp
   2 amino acids  =  160.2 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Neisseria meningitidis Type
II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D16A,
D587A, H588A, and N611A
NmdCas9
3019 .. 6261  =  3243 bp
1081 amino acids  =  124.0 kDa
   Segment 5:  
   4780 .. 4845  =  66 bp
   22 amino acids  =  2.5 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Neisseria meningitidis Type
II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D16A,
D587A, H588A, and N611A
NmdCas9
3019 .. 6261  =  3243 bp
1081 amino acids  =  124.0 kDa
   Segment 6:  
   4846 .. 4848  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Neisseria meningitidis Type
II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D16A,
D587A, H588A, and N611A
NmdCas9
3019 .. 6261  =  3243 bp
1081 amino acids  =  124.0 kDa
   Segment 7:  
   4849 .. 6261  =  1413 bp
   471 amino acids  =  54.7 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Neisseria meningitidis Type
II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D16A,
D587A, H588A, and N611A
NmdCas9
3019 .. 6261  =  3243 bp
1081 amino acids  =  124.0 kDa
7 segments
Product: catalytically dead mutant of the Cas9
endonuclease from the Neisseria meningitidis Type
II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D16A,
D587A, H588A, and N611A
AmpR
9718 .. 10,578  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   9718 .. 9786  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9718 .. 10,578  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   9787 .. 10,578  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9718 .. 10,578  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
superfolder GFP
6313 .. 7026  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
6313 .. 7026  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
7045 .. 7758  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
7045 .. 7758  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
7777 .. 8490  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
superfolder GFP
7777 .. 8490  =  714 bp
238 amino acids  =  26.8 kDa
Product: GFP variant that folds robustly even when
fused to poorly folded proteins (Pédelacq et al.,
2006)
LTR
1 .. 634  =  634 bp
long terminal repeat (LTR) from HIV-1
LTR
1 .. 634  =  634 bp
long terminal repeat (LTR) from HIV-1
WPRE
8512 .. 9100  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
8512 .. 9100  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
10,749 .. 11,337  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
10,749 .. 11,337  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
2299 .. 2602  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2299 .. 2602  =  304 bp
human cytomegalovirus immediate early enhancer
RRE
1303 .. 1536  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1303 .. 1536  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
LTR (ΔU3)
9175 .. 9408  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
LTR (ΔU3)
9175 .. 9408  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
CMV promoter
2603 .. 2806  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
2603 .. 2806  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
2058 .. 2175  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2058 .. 2175  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
9613 .. 9717  =  105 bp
AmpR promoter
9613 .. 9717  =  105 bp
SV40 NLS
2986 .. 3006  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
2986 .. 3006  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6280 .. 6300  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6280 .. 6300  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
tet operator
2808 .. 2826  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
2808 .. 2826  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
2829 .. 2847  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
2829 .. 2847  =  19 bp
bacterial operator O2 for the tetR and tetA genes
ATG
2980 .. 2982  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
2980 .. 2982  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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